The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. A key enzyme in this cycle, vitamin K epoxide reductase (VKOR), is the target of vitamin K antagonists (VKAs). Despite their extensive clinical use, the dose of VKAs (e.g., warfarin) is hard to regulate and overdose can lead to fatal bleeding. Improving the dose regulation requires understanding how VKAs inhibit VKOR, which is a membrane- embedded enzyme that is difficult to characterize with structural and biochemical studies. Our long-term goal is to elucidate the physiological process of the entire vitamin K cycle and its interaction with VKAs. This cycle begins with ?-carboxylation, a modification required for the activity of vitamin-K-dependent proteins, including several coagulation factors. The carboxylase activity requires the epoxidation of vitamin K hydroquinone. VKOR regenerates this cofactor by reducing the epoxide, and this reductase activity is maintained by electron-transfer pathways. VKOR also has a paralog, VKORL, which has the same activity but is relatively insensitive to warfarin inhibition. Owing to differences in tissue distribution, VKOR primarily supports blood coagulation and VKORL likely supports non-coagulation processes. The objective of this application is to elucidate the mechanisms of VKOR and VKORL catalysis and vitamin K antagonism using our expertise in membrane structure biology. Our hypotheses are: (1) the narrow therapeutic window of warfarin is in part because it is a tight-binding inhibitor whose dose range is limited by VKOR levels; (2) the cellular activity of VKOR is maintained by alternative electron-transfer pathways; and (3) a common structural mechanism governs the warfarin insensitivity of VKORL and warfarin-resistant mutations in VKOR. To support these hypotheses, we have achieved a long-standing goal of determining the crystal structures of human VKOR with several VKAs and with the substrate, vitamin K epoxide, and have determined the structures of a VKORL homolog in its warfarin-bound and ligand-free states. We found distinct groups of warfarin-resistant mutations in VKOR, and identified key residues that control the warfarin sensitivity of VKORL. We also showed that warfarin is a tight-binding inhibitor in vitro. We will test our hypotheses with three specific aims: (1) we will show the tight binding of warfarin in a cellular environment, understand its correlation with VKOR's redox status, and test whether reducing VKOR can release bound warfarin; (2) we will determine how VKAs inhibit VKOR catalysis, elucidate the reduction steps and reaction intermediate of VKOR, and characterize the electron-transfer pathways that maintain VKOR activity; and 3) we will define the structural basis of warfarin resistance and investigate whether VKORL variations lead to osteoporosis. Armed with our recently developed structural tools, we will demonstrate innovative concepts about the inhibition range of VKAs, the catalytic pathway of VKOR, and mutations interfering with coagulation and bone health. Thus, the proposed studies will significantly advance our knowledge of VKOR function and its interaction with VKAs, leading to improved warfarin management.